Ex Vivo Antisense Oligonucleotides to Proliferating Cell Nuclear Antigen and Cdc2 Kinase Inhibit Graft Coronary Artery Disease

Abstract

Background —The long-term success of cardiac transplantation is limited by graft coronary artery disease (GCAD). Antisense oligonucleotides (ASs) to proliferating cell nuclear antigen (PCNA) and Cdc2 kinase (Cdc2 k) can arrest cell cycle progression and inhibit neointimal hyperplasia. Transforming growth factor-β 1 (TGF-β 1 ) has been implicated in vascular smooth muscle cell (VSMC) activation. The role of TGF-β 1 in GCAD remains unclear. We hypothesized that ASs to PCNA and Cdc2 k would inhibit VSMC proliferation and GCAD. Methods and Results —In vitro VSMC proliferation was determined after pretreatment with AS solution or medium alone followed by angiotensin II stimulation. PVG-to-ACI rat heterotopic cardiac transplantation procedures were performed after ex vivo pressure-mediated transfection of ASs to PCNA and Cdc2k or saline alone. At postoperative days 30, 60, and 90, allografts were assessed for GCAD, percent neointimal macrophages and VSMCs, and TGF-β 1 activity. AS pretreatment significantly attenuated VSMC proliferation. At postoperative day 90, percent affected arteries, percent occlusion, and intima-media ratio demonstrated severe GCAD in saline-treated allografts, whereas these parameters were significantly lower in AS-treated allografts. Percent neointimal macrophages and VSMCs was reduced in AS-treated allografts. TGF-β 1 activity was increased in saline compared with AS-treated allografts and nontransplanted heart controls. Conclusions —ASs to PCNA and Cdc2 k inhibit VSMC proliferation in vitro and reduce GCAD, percent neointimal VSMCs and macrophages, and TGF-β 1 activity in vivo. TGF-β 1 may play a “response to injury” role in the development of GCAD. The prevention of GCAD via AS inhibition of cell cycle regulatory genes before reperfusion may offer a useful clinical alternative to current therapeutic strategies.